In the yeast Saccharomyces cerevisiae, a system has been designed to study the effect of defined DNA double-strand breaks (DSB) in plasmids on recombination and repair at various chromosomal sites. In this system, a diploid yeast strain with a set of heteroallelic markers harbors two plasmids: a low copy CEN plasmid that carries a gene coding for the site-specific endonuclease HO under the control of the inducing promoter GAL, a high copy 2mu plasmid that carries a site at which the HO endonuclease cuts. Since the chromosomal HO-cut sites are deleted in this strain, derepression of HO should generate a defined multiple DSB only at HO-cut sites carried by the 2 mu plasmids. Preliminary experiments indicate that the depression of the HO gene and subsequent DSBs in plasmids causes induction of recombination in the chromosome. The mechanism of this trans-acting effect of DSBs is being investigated in terms of effects on survival, mutation, and recombination.